Diabetes mellitus is a complex and life threatening disease that has been known for more than 2000 years. It occurs in mammals as diverse as monkeys, dogs, rats, mice and human beings. The discovery of insulin and its purification in 1921 for use in people provided a partial treatment for diabetes that is still in widespread use today. Insulin levels are ordinarily adjusted by the body on a moment to moment basis to keep the blood sugar level within a narrow physiological range. Periodic insulin injections, however, can only approximate the normal state because the cellular response to insulin in many cases is also reduced. Consequently, for these and other reasons which will be discussed in detail below, life threatening complications still occur during the lifetime of treated diabetic patients, especially in the case of type 2 (adult-onset) diabetes.
Diabetes arises from various causes, including dysregulated glucose sensing or insulin secretion (Maturity onset diabetes of youth; MODY), autoimmune-mediated β-cell destruction (type 1), or insufficient compensation for peripheral insulin resistance (type 2). (Zimmet, P. et al., Nature 414:782-787 (2001)). Type 2 diabetes is the most prevalent form of the disease: It is closely associated with obesity, usually occurs at middle age, and afflicts 18.2 million Americans. The common mechanism causing peripheral insulin resistance and β-cell failure is important to understand.
Peripheral insulin resistance contributes to type 2 diabetes, but β-cell failure is an essential feature of all types of diabetes. β-cells frequently fail to compensate for insulin resistance, apparently because the IRS2-branch of the insulin and IGF signaling cascade which mediates insulin signaling in target tissues also is essential for β-cell growth, function and survival. (Withers, D. J. et al., Nature 391:900-904 (1998)).
Because insulin resistance is a cause of metabolic dysregulation and diabetes, understanding its molecular basis is an important goal. Genetic mutations are obvious sources of life-long insulin resistance, but they are associated with rare metabolic disorders and thus difficult to identify in the general population. Inflammation is associated with insulin resistance and provides a framework to understand how diet, acute or chronic stress, and obesity might cause insulin resistance. Proinflammatory cytokines, including IL6 and tumor necrosis factor-α (TNFα) that are secreted from leukocytes during inflammation, are also produced in adipose tissue. TNFα promotes serine phosphorylation of IRS-proteins, which correlates closely with insulin resistance. (Hotamisligil, G. S. et al., Science 259:87-91 (1999); Hotamisligil, G. S. et al., Science 271:665-668 (1996); Peraldi, P. et al., J. Biol. Chem. 271:13018-13022 (1996)). Although TNFα regulates various kinases, the NH2-terminal Jun kinase (Jnk) is a prominent effector because it binds to IRS1 and IRS2 and phosphorylates serine residues that inhibit the interaction between IRS1 and the insulin receptor. (Aguirre, V. et al., J. Biol. Chem. 277:1531-1537 (2002)). The knockout of Jnk1 in obese mice, or general inhibition of serine kinases by high doses of salicylates reduces Ser phosphorylation of IRS1 and reverses hyperglycemia, hyperinsulinemia, dyslipidemia in obese rodents by sensitizing insulin signaling pathways. (Fruebis, J. et al., Proc. Natl. Acad. Sci. U.S.A 98:2005-2010 (2001); Yuan, M. et al., Science 293:1673-1677 (2001)).
Ubiquitin-mediated degradation of IRS-proteins also promotes insulin resistance (FIG. 1). IL6 secreted from leukocytes and adipocytes increases expression of SOCS1 and SOCS3, known for the ability to suppress cytokine signaling. Another function of SOCS1 and SOCS3 is to recruit an elongin BC-based ubiquitin ligase into the IRS-protein complex to mediate ubiquitinylation. Thus, ubiquitin-mediated degradation of IRS-proteins might be a general mechanism of cytokine-induced insulin resistance that contributes to diabetes or β-cell failure. (Krebs, D. L. et al., Sci. STKE. 2003, E6 (2003)). Modern genomic approaches have revealed new cytokines secreted directly from adipocytes that directly influence nutrient homeostasis and insulin sensitivity, including leptin, adiponectin, resistin and others that will reveal new mechanisms to modulate insulin sensitivity.
The activity of protein or lipid phosphatases, including PTP1B, SHIP2 or pTEN modulates insulin sensitivity (FIG. 1). Disruption of each of these genes in mice increases insulin sensitivity, suggesting that each might be a target for inhibitor design. PTP1B resides in the endoplasmic reticulum where it dephosphorylates the insulin receptor during internalization and recycling to the plasma membrane. (Haj, F. G. et al., Science 295:1708-1711 (2002)). This specialized mechanism appears to limit unwanted side effects associated with inhibition of phosphatases, including unregulated cell growth.
The insulin receptor is the prototype for a family of homologous integral membrane proteins composed of an extracellular insulin-binding domain that controls the activity of an intracellular tyrosine kinase. A 150-kb gene on chromosome 19 composed of 22 exons encodes the human proreceptor. During translation, two homologous proreceptors form a disulfide-linked dimer that is glycosylated and cleaved to form a heterotetramer of two extracellular α-subunits and two trans-membrane β-subunits. Insulin binds to the juxtaposed α-subunits facilitating ATP binding and tyrosine autophosphorylation of the β-subunit, which activates the kinase and recruits cellular substrates to initiate signal transduction. The full insulin-signaling pathway as presently known is summarized in the STKE Connections Map. (White, M., Insulin Signaling Pathway, Sci. STKE Connections Map, as seen November 2003, http://stke.sciencemag.org/cgi/cm/CMP—12069).
Selective insulin binding is complicated by tissue-specific alternative splicing that directs synthesis of two insulin receptor isoforms (IRa and IRb), and by post-translational assembly of hybrids between these isoforms and the homologous IGF1 receptor (IGF1R). (Frasca, F. et al., Mol. Cell Biol. 19:3278-3288 (1999)). IRb exclusively binds insulin, whereas IRa binds both insulin and IGF2 with similar affinities: Dysregulated splicing alters fetal growth patterns and contributes to rare forms of insulin resistance in adults. (Frasca, 1989; Savkur, R. S. et al., Nat. Genet. 29:40-47 (2001)) Moreover, hybrid receptors composed of an αβ-dimer from the IGF1R and the IRb selectively bind IGF1, whereas hybrid receptors composed of IGF1R and IRa bind IGFs and insulin with similar affinities (FIG. 1).
The first member of the insulin receptor substrate family of proteins was discovered in 1985, and subsequent research efforts revealed the existence of related IRS family members as well as the signaling pathways to which the IRS proteins are linked. After the discovery that the Insulin Receptor (IR) possessed a tyrosine kinase enzyme activity, many groups searched for insulin receptor substrates that might regulate downstream signaling from the receptor. The first evidence for the existence of an actual target protein for the Insulin Receptor, subsequently named an Insulin Receptor Substrate, or “IRS” protein, resulted from the use of phosphotyrosine antibody immunoprecipitates which surprisingly revealed a 185-kDa phosphoprotein (pp185) in insulin-stimulated hepatoma cells. (White, M. F. et al., Nature 318:183-186 (1985)). Purification and molecular cloning of pp185 revealed one of the first signaling scaffolds as well as the first Insulin Receptor Substrate protein (IRS1). (U.S. Pat. No. 5,260,200; Sun, X. J. et al., Nature 352:73-77 (1991)). IRS1 was determined to be biologically important because it was phosphorylated immediately after insulin stimulation, and catalytically active insulin receptor mutants that failed to phosphorylate IRS1 were biologically inactive. Most, if not all, insulin signals are produced or modulated through tyrosine phosphorylation of IRS1, IRS2 or its homologs; or other scaffold proteins including SHC, CBL, APS and SH2B, GAB1, GAB2, DOCK1, and DOCK2. Although the role of each of these substrates merits attention, work with transgenic mice suggests that many insulin responses, especially those that are associated with somatic growth and carbohydrate metabolism, are mediated through IRS1 or IRS2.
IRS-proteins are composed of multiple interaction domains and phosphorylation motifs, but appear to lack intrinsic catalytic activities. All IRS-proteins contain an NH2-terminal pleckstrin homology (PH) domain adjacent to a phosphotyrosine-binding (PTB) domain, followed by a COOH-terminal tail with numerous tyrosine and serine phosphorylation sites. The PTB domain binds directly to the phosphorylated NPXY-motif—Asn-Pro-Xaa-Tyr(Pi), Xaa represents any amino acid—in the activated receptors for insulin, IGFs or interleukin-4 (IL4); the PH domain also couples IRS-proteins to activated receptors, but the mechanism is unclear. (Yenush, L. et al., Mol. Cell Biol. 18:6784-6794 (1998)). Other receptors also recruit and phosphorylate IRS-proteins, including those for growth hormone, IL-9, IL-13 and IL-15, and various integrins. (Shaw, L. M., Mol. Cell Biol. 21:5082-5093 (2001)).
Tyrosine phosphorylation sites in IRS1 and IRS2 bind common effector proteins, including enzymes (phosphoinositide 3-kinase, the phosphatase SHP2, or the tyrosine kinase fyn) or adapters (SOCS1, SOCS3, GRB2, NCK, CRK, SHB and others).
Activation of PI3K during association with IRS proteins increases the activity of protein kinase B (PKB), which phosphorylates various substrates including BAD (important for cell survival), GSK3β (regulating growth and glycogen synthesis), and Foxo1 (controlling gene expression) (FIG. 1). A role for Foxo1 in insulin or IGF action was revealed by mutations in the C. elegans ortholog Daf16. (Ogg, S. et al., Nature 389:994-999 (1997)). During insulin or IGF stimulation, Daf16 and Foxo1 are phosphorylated by PKB and accumulate in the cytosol. Nuclear exclusion of Foxo1 inhibits hepatic gluconeogenesis, but stimulates adipocyte differentiation and pancreatic β-cell function. (Nakae, J. et al., Dev. Cell 4:119-129 (2003); Kitamura, T. et al., J. Clin. Invest 110:1839-1847 (2002); Puigserver, P. et al., Nature 423:550-555 (2003)).
IRS1 contains many tyrosine phosphorylation sites that are phosphorylated during insulin and insulin-like growth factor 1 (IGF1) stimulation, and bind to the Src homology-2 domains in various signaling proteins. The interaction between IRS1 and p85 activates the class 1A phosphotidylinositide 3-kinase, thereby revealing the first insulin signaling cascade that could be reconstituted successfully in cells and test tubes.
Several experiments suggested that other related proteins might exist: IRS1 antibodies did not react completely with the phosphotyrosine containing protein that migrated at 185 kDa during SDS-PAGE; FDCP1 cells contained a protein with characteristics similar to those of IRS1 but failed to react with antibodies directed against IRS1; the liver of transgenic mice lacking IRS1 still contained a protein in liver that had characteristics of IRS1. All of these finding led to the purification and cloning of Insulin Receptor Substrate 2 (IRS2), a second member of the IRS family. (U.S. Pat. No. 5,858,701; Sun, X. J. et al., Nature 377:173-177 (1995)).
Experiments in transgenic mice revealed involvement of IRS1 and IRS2 in promoting somatic growth and nutrient homeostasis. Without IRS1, mice are 50% smaller than normal from birth until they die at 2 years of age. Mice without IRS1 have less body fat and are glucose intolerant. In mice, IRS2 is important for peripheral insulin action, as mice lacking IRS2 display glucose intolerance and hyperlipidemia.
Disruption of the IRS2 gene in mice using standard gene knockout approaches results in diabetes that develops during the first 10 to 12 weeks of age. Pancreatic β-cells are lost from these mice as they age, and genes that are important for β-cell function are down regulated in mice lacking IRS2.